1. Field of the Invention
The invention relates to luminescent dyes, including reactive dyes and dye conjugates; and their biological uses.
2. Description of Related Art
Luminescent probes are valuable reagents for the analysis and separation of molecules and cells and for the detection and quantification of other materials. A very small number of luminescent molecules can be detected under optimal circumstances. Some specific examples of the application of fluorescent probes are (1) identification and separation of subpopulations of cells in a mixture of cells by the techniques of fluorescence flow cytometry, fluorescence-activated cell sorting and fluorescence microscopy; (2) determination of the concentration of a substance that binds to a second species (e.g., antigen-antibody reactions) in the technique of fluorescence immunoassay; (3) localization of substances in gels and other insoluble supports by the techniques of fluorescence staining. These techniques are described by Herzenberg, et al., “CELLULAR IMMUNOLOGY” 3rd ed., Chapter 22; Blackwell Scientific Publications (1978); and by Goldman, “FLUORESCENCE ANTIBODY METHODS”, Academic Press, New York, (1968); and by Taylor, et al., APPLICATIONS OF FLUORESCENCE IN THE BIOMEDICAL SCIENCES, Alan Liss Inc., (1986), and Shapiro, PRACTICAL FLOW CYTOMETRY, 4th ed., Wiley-Liss (2003), all of which are incorporated herein by reference.
When employing luminescent dyes for the above purposes, there are many constraints on the choice of the fluorescent dye. One constraint is the absorption and emission characteristics of the fluorescent dye, since many ligands, receptors, and materials in the sample under test, e.g. blood, urine, cerebrospinal fluid, will fluoresce and interfere with an accurate determination of the fluorescence of the fluorescent label. This phenomenon is called autofluorescence or background fluorescence. Another consideration is the ability to conjugate the fluorescent dye to ligands and receptors and other biological and non-biological materials and the effect of such conjugation on the fluorescent dye. In many situations, conjugation to another molecule may result in a substantial change in the fluorescent characteristics of the fluorescent dye and, in some cases, substantially destroy or reduce the quantum efficiency of the fluorescent dye. It is also possible that conjugation with the fluorescent dye will inactivate the function of the molecule that is labeled. A third consideration is the quantum efficiency of the luminescent dyes which should be high for sensitive detection. A fourth consideration is the light absorbing capability, or extinction coefficient, of the luminescent dyes, which should also be as large as possible. Also of concern is whether the fluorescent molecules will interact with each other when in close proximity, resulting in self-quenching. An additional concern is whether there is non-specific binding of the luminescent dyes to other compounds or container walls, either by themselves or in conjunction with the compound to which the fluorescent dye is conjugated.
The applicability and value of the methods indicated above are closely tied to the availability of suitable fluorescent compounds. In particular, there is a need for fluorescent substances that emit in the longer wavelength region (yellow to near infrared), since excitation of these chromophores produces less autofluorescence and also multiple chromophores fluorescing at different wavelengths can be analyzed simultaneously if the full visible and near infrared regions of the spectrum can be utilized. Fluorescein, a widely used fluorescent compound, is a useful emitter in the green region although in certain immunoassays and cell analysis systems background autofluorescence generated by excitation at fluorescein absorption wavelengths limits the detection sensitivity. However, the conventional red fluorescent label rhodamine has proved to be less effective than fluorescein.
Phycobiliproteins have made an important contribution because of their high extinction coefficient and high quantum yield. These chromophore-containing proteins can be covalently linked to many proteins and are used in fluorescence antibody assays in microscopy and flow cytometry. The phycobiliproteins have the disadvantages that (1) the protein labeling procedure is relatively complex; (2) the protein labeling efficiency is not usually high (typically an average of 0.5 phycobiliprotein molecules per protein); (3) the phycobiliproteins are natural products and their preparation and purification are complex; (4) the phycobiliproteins are expensive; (5) there are at present no phycobiliproteins available as labeling reagents that fluoresce further to the red region of the spectrum than allophycocyanine, which fluoresces maximally at 680 nm; (6) the phycobiliproteins are large proteins with molecular weights ranging from 33,000 to 240,000 and are larger than many materials that are desirable to label, such as metabolites, drugs, hormones, derivatized nucleotides, and many proteins including antibodies. The latter disadvantage is of particular importance because antibodies, avidin, DNA-hybridization probes, hormones, and small molecules labeled with the large phycobiliproteins may not be able to bind to their targets because of steric limitations imposed by the size of the conjugated complex.
Other techniques involving histology, cytology, immunoassays would also enjoy substantial benefits from the use of a fluorescent dye with a high quantum efficiency, absorption and emission characteristics at longer wavelengths, having simple means for conjugation and being substantially free of nonspecific interference.
Fluorescent compounds are covalently or noncovalently attached to other materials to impart color and fluorescence. Brightly luminescent dyes permit detection or location of the attached materials with great sensitivity. Certain carbocyanine dyes have demonstrated utility as labeling reagents for a variety of biological applications, e.g. U.S. Pat. No. 4,981,977 to Southwick, et al. (1991); U.S. Pat. No. 5,268,486 to Waggoner, et al. (1993); U.S. Pat. No. 5,569,587 to Waggoner (1996); U.S. Pat. No. 5,569,766 to Waggoner, et al. (1996); U.S. Pat. No. 5,486,616 to Waggoner, et al. (1996); U.S. Pat. No. 5,627,027 to Waggoner (1997); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S. Pat. No. 5,877,310 to Reddington, et al. (1999); U.S. Pat. No. 6,002,003 to Shen, et al. (1999); U.S. Pat. No. 6,004,536 to Leung, et al. (1999); U.S. Pat. No. 6,008,373 to Waggoner, et al. (1999); U.S. Pat. No. 6,043,025 to Minden, et al. (2000); U.S. Pat. No. 6,127,134 to Minden, et al. (2000); U.S. Pat. No. 6,130,094 to Waggoner, et al. (2000); U.S. Pat. No. 6,133,445 to Waggoner, et al. (2000). Nevertheless, many carbocyanine dyes are known to share certain disadvantages, e.g. severe quenching of the fluorescence of carbocyanine dyes in biopolymer conjugates, e.g. quenching of Cy5 and Cy7 dye variants on conjugates, as discussed by Gruber, et al., BIOCONJUGATE CHEM., 11, 696 (2000). In addition, certain desired sulfoalkyl derivatives of the reactive carbocyanine dyes are difficult to prepare, as indicated for Cy3 and Cy5 variants by Waggoner and colleagues in BIOCONJUGATE CHEM., 4, 105, 109 (1993). Cyanine dyes also have a very strong tendency to self-aggregate (i.e. stack), which can significantly reduce the fluorescence quantum yields, as described in the extensive review by Mishra, et al., CHEM. REV., 100, 1973 (2000).
Another problem with the existing carbocyanine labeling dyes is the free rotation/vibration of two indolium (or benzothiazolium, or benzoimidazolium) heads around the middle conjugated double bonds that significantly reduce their fluorescence intensities. This phenomenon is called ‘loose belt effect’ that is described in “MODERN MOLECULAR PHOTOCHEMISTRY”, Chapters 5 and 6, University Science Books, Sausalito, Calif., authored by Nicholas J. Turro (1991).
This so-called ‘loose belt effect’ can be eliminated by the crosslinking of the two heads. 1,1′-crosslinking of cyanines is disclosed by R. Singh, et al. WO 01/02374 (2001), which is supposed to eliminate the ‘loose belt effect’ described above. However, Diwu et al., U.S. Pat. No. 7,465,810, observed that the 1,1′-crosslinking actually caused the decreased fluorescence quantum yield of dye-protein conjugates compared to that of non-crosslinked carbocycanine-protein conjugates at the similar ratios of dye/protein. This unfavorable fluorescence decrease might be caused by the inappropriate stereochemistry of 1,1′-crosslinking or by the increased hydrophobicity, which results from the addition of the highly hydrophobic bridge.
Diwu et al., U.S. Pat. No. 7,465,810, observed that the fluorescence properties of cyanine labeling dyes were improved by the intramolecularly crosslinking of the two indoline moieties between the 1-position and 3′-position. The 1,3′-crosslinked carbocyanines are superior to those of conjugates of spectrally similar 1,1′-crosslinked or non-crosslinked dyes. This improvement with 1,3′-cros slinked cyanine might result from the favorable configuration. However, the hydrophobic bridge significantly increases the dye hydrophobicity. Compared to the non-crosslinked carbocyanine dyes the conjugation of either 1,3′-crosslinked or 1,1′-crosslinked carbocyanines to proteins and other biological molecules often caused the precipitation of the labeling target molecules due to the significantly reduced water solubility of labeling dyes caused by the highly hydrophobic cross linker. In some cases, the cros slinking of carbocyanines by a highly hydrophobic bridge result in a complete activity loss of the target molecules to be labeled.
We discovered new water-soluble cross linkers that do not only eliminate the ‘loose belt effect’ to increase the fluorescence quantum yields of labeling dyes, but also increase their water solubility, thus eliminating the drawback of protein precipitation with the existing luminescent dyes that contain a hydrophobic cross linking bridge. We found that this strategy of water-soluble bridge can also be used improve the labeling and spectral properties of luminescent dyes of other types.
Xanthene dyes are another class of fluorescent probes that are predominantly used for labeling proteins and other biological molecules, e.g., U.S. Pat. No. 7,704,284 to Eliu, et al. (2010); U.S. Pat. No. 7,491,830 to Lam, et al. (2009); U.S. Pat. No. 7,344,701 to Reddington, et al. (2008); U.S. Pat. No. 6,229,055 to Klaubert, et al. (2001); U.S. Pat. No. 6,130,101 to Mao et al. (2000). We discovered that the ‘loose belt effect’ of xanthene dyes can be eliminated by the crosslinking of the two substitute heads with the new water-soluble cross linkers while their water solubilities are also significantly increased. The same principle can also be used to simultaneously increase the fluorescence quantum yields and water solubility of oxazole dye-protein conjugates and luminescent metal complex-protein conjugates.